Endocannabinoids are endogenously produced fatty acid derivatives that act at the cannabinoid 1 and 2 receptors. The two predominant endocannabinoids are 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (AEA). These molecules are involved in a plethora of biological processes and, in particular, have been shown to modulate analgesic activity. AEA and 2-AG are synthesized on demand by phospholipases and recent work by the Marnett lab has shown that 2-AG and AEA are both substrates of cyclooxygenase-2 (COX-2). Importantly, the inhibition of COX-2 by several non-steroidal anti-inflammatory drugs (NSAIDs), in particular the (S)-2-arylpropionic acid derivative NSAIDs, has been shown to be more potent with respect to the inhibition of endocannabinoid oxygenation as compared to the inhibition of arachidonic acid (AA) oxygenation. The (R)-enantiomers of 2-arylpropionic acid derivative NSAIDs have long been classified as non-inhibitors of COX-2 since they do not inhibit the oxygenation of AA by COX-2. However, our recent work indicates that they are potent inhibitors of endocannabinoid oxygenation by COX-2. Of particular interest, the most potent (R)-enantiomer of a 2- arylpropionic acid derivative NSAID, (R)-flurbiprofen, has been shown to provide analgesia in neuropathic pain models by increasing the levels of AEA. The observed substrate-selective inhibition of COX-2 by (R)-flurbiprofen offers a novel mechanism for achieving this increased AEA level and corresponding analgesia. The mechanism by which (R)-flurbiprofen effects substrate-selective inhibition of COX-2 is presently unknown and will be elucidated using a combination of site-directed mutagenesis and X-ray crystallography. Site-directed mutagenesis will be used to create binding site mutants to probe for residues critical to the substrate-selective inhibition of COX-2 by (R)-flurbiprofen. The mechanism of substrate-selective inhibition by (R)-flurbiprofen will also be defined by solving the crystal structure of (R)-flurbiprofen bound to murine COX-2. The structural and functional insights into the mechanism of substrate-selective inhibition of COX-2 provided by the site-directed mutagenesis and X- ray crystallography will be used to design and synthesize (R)-flurbiprofen analogs with improved potency. The synthesized analogs will be evaluated for substrate-selective inhibition by using an in vitro assay with purified murine or human COX-2 followed by ex vivo testing using RAW 264.7 macrophages and primary dorsal root ganglia as model systems. The most potent substrate-selective inhibitors will then be evaluated and compared to (R)-flurbiprofen in vivo using the mouse carrageenan footpad inflammation model. By defining the mechanism of substrate-selective inhibition of COX-2 by (R)- flurbiprofen and developing and testing novel inhibitors, the project will elucidate a novel mechanism of endocannabinoid regulation and develop lead compounds for the treatment of neuropathic pain.